Fluorescent Polymer Dots, Methods For Their Preparation, And Uses Thereof

Abstract

There is provided a fluorescent polymer dot (FPdot) comprising one or more fluorescent conjugated polymers forming a hydrophobic core and one or more amphiphilic molecules, each amphiphilic molecule comprising a hydrophobic end embedded in the core and a hydrophilic end that forms a hydrophilic shell surrounding the core, wherein said hydrophobic end comprises one or more aliphatic chain moieties. There is also provided a method for preparing the fluorescent polymer dot comprising preparing a mixture of conjugated polymer and amphiphilic molecule in an aprotic solvent; and adding said mixture to a protic solvent. The fluorescent polymer dots may be useful in biomedical imaging applications.

1 . A fluorescent polymer dot comprising one or more fluorescent conjugated polymers, forming a hydrophobic core and one or more amphiphilic molecules, each amphiphilic molecule comprising a hydrophobic end embedded in the core and a hydrophilic end that forms a hydrophilic shell surrounding the core, wherein said hydrophobic end comprises one or more aliphatic chain moieties. 
     
     
         2 . The fluorescent polymer dot according to  claim 1 , wherein the one or more aliphatic chain moieties are saturated. 
     
     
         3 . The fluorescent polymer according to  claim 2 , wherein the one or more saturated aliphatic chain moieties comprises 10 to 24 carbon atoms. 
     
     
         4 . The fluorescent polymer dot according to any one of  claims 2  to  3 , wherein the one or more saturated aliphatic chain moieties are part of a lipid moiety. 
     
     
         5 . The fluorescent polymer dot according to  claim 4 , wherein the lipid moiety is selected from the group consisting of 1,2-distearoyl-sn-glycero-3-phosphoethanolamino (DSPE) moiety, 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE) moiety, 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE) moiety, and (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine) (DPPE) moiety. 
     
     
         6 . The fluorescent polymer dot according to any one of  claims 1  to  5 , wherein the hydrophilic end of the amphiphilic molecule comprises one or more polymers selected from the group consisting of polyoxyalkylene, polyalkylene glycol, and polycarboxyalkylene. 
     
     
         7 . The fluorescent polymer dot according to any one of  claims 1  to  6 , wherein the amphiphilic molecule comprises one or more polymers selected from the group consisting of polyethylene glycol (PEG), polypropylene glycol, polybutylene glycol, polycarboxymethylene, polycarboxyethylene (or polyacrylic acid (PAA)), polycarboxypropylene, and polycarboxybutylene. 
     
     
         8 . The fluorescent polymer dot according to any one of  claims 1  to  7 , wherein the hydrophilic end of the amphiphilic molecule is attached to one or more functional groups. 
     
     
         9 . The fluorescent polymer dot according to  claim 8 , wherein the functional group is selected from the group consisting of alkoxy, amino, carboxy, imide, and biotin. 
     
     
         10 . The fluorescent polymer dot according to any one of  claims 1  to  9 , wherein the amphiphilic molecule is selected from the group consisting of:
 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-2000] (DSPE-PEG); 
 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG-OCH 3 ), 
 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG-NH 2 ); 
 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000] (DSPE-PEG-COOH); 
 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] (DSPE-PEG-maleimide); 
 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-2000] (DSPE-PEG-biotin); 
 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-2000] (DMPE-PEG); 
 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DMPE-PEG-OCH 3 ); 
 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DMPE-PEG-NH 2 ); 
 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000] (DMPE-PEG-COOH); 
 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] (DMPE-PEG-maleimide); 
 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-2000] (DMPE-PEG-Biotin); 
 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-2000] (DLPE-PEG); 
 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DLPE-PEG-OCH 3 ); 
 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DLPE-PEG-NH 2 ); 
 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000] (DLPE-PEG-COOH); 
 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] (DLPE-PEG-maleimide)-; 
 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-2000] (DLPE-PEG-Biotin); 
 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-2000] (DPPE-PEG); 
 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DPPE-PEG-OCH 3 ); 
 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DPPE-PEG-NH 2 ); 
 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000] (DPPE-PEG-COOH); 
 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] (DPPE-PEG-maleimide); and 
 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-2000] (DPPE-PEG-Biotin); 
 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[(polyacrylic acid)] (DSPE-PAA); 
 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyacrylic acid)] (DSPE-PAA-OCH 3 ), 
 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyacrylic acid)] (DSPE-PAA-NH 2 ); 
 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyacrylic acid)] (DSPE-PAA-COOH); 
 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyacrylic acid)] (DSPE-PAA-maleimide); 
 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyacrylic acid)] (DSPE-PAA-biotin); 
 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[(polyacrylic acid)] (DMPE-PAA); 
 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyacrylic acid)] (DMPE-PAA-OCH 3 ); 
 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyacrylic acid)] (DMPE-PAA-NH 2 ); 
 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyacrylic acid)] (DMPE-PAA-COOH); 
 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyacrylic acid)] (DMPE-PAA-maleimide); 
 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyacrylic acid)] (DMPE-PAA-Biotin); 
 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[(polyacrylic acid)] (DLPE-PAA); 
 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyacrylic acid)] (DLPE-PAA-OCH 3 ); 
 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyacrylic acid)] (DLPE-PAA-NH 2 ); 
 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyacrylic acid)] (DLPE-PAA-COOH); 
 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyacrylic acid)] (DLPE-PAA-maleimide); 
 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyacrylic acid)] (DLPE-PAA-Biotin); 
 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[(polyacrylic acid)] (DPPE-PAA); 
 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyacrylic acid)] (DPPE-PAA-OCH 3 ); 
 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyacrylic acid)] (DPPE-PAA-NH 2 ); 
 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyacrylic acid)] (DPPE-PAA-COOH); 
 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyacrylic acid)] (DPPE-PAA-maleimide); and 
 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyacrylic acid)] (DPPE-PAA-Biotin). 
 
     
     
         11 . The fluorescent polymer dot according to any one of  claims 1  to  10 , wherein the fluorescent conjugated polymer comprises repeating units of optionally substituted cyclic group linked to O-containing heteroaryl group. 
     
     
         12 . The fluorescent polymer dot according to  claim 11 , wherein the optionally substituted cyclic group is an optionally substituted monocyclic or polycyclic group. 
     
     
         13 . The fluorescent polymer dot according to  claim 12 , wherein the optionally substituted monocyclic or polycyclic group is aromatic. 
     
     
         14 . The fluorescent polymer dot according to any one of  claims 11  to  13 , wherein the O-containing heteroaryl group is monocyclic or polycyclic. 
     
     
         15 . The fluorescent polymer dot according to any one of  claims 11  to  14 , wherein the O-containing heteroaryl group is selected from the group consisting of diazole, benzodiazole, oxazole, benzooxazole, pyran, furan, and benzofuran. 
     
     
         16 . The fluorescent polymer dot according to any one of  claims 11  to  15 , wherein the fluorescent conjugated polymer is poly(9,9-dihexylfluorene-alt-2,1,3-benzoxadiazole) (PFBD). 
     
     
         17 . The fluorescent polymer dot according to any one of  claims 1  to  16 , wherein a biological molecule is conjugated to the surface of said dot. 
     
     
         18 . The fluorescent polymer dot according to any one of  claims 8  to  17 , wherein a biological molecule is conjugated to the surface of the dot via the functional group. 
     
     
         19 . The fluorescent polymer dot according to any one of  claim 17  or  18 , wherein the biological molecule is selected from the group consisting of amino acids, peptides, polypeptides, nucleic acids, carbohydrates, lipids, fatty acids, antibodies, aptamers, and proteins. 
     
     
         20 . The fluorescent polymer dot according to any one of  claims 17  to  19  wherein the biological molecule is a cell penetrating peptide. 
     
     
         21 . The fluorescent polymer dot according to  claim 20 , wherein the cell penetrating peptide is a Tat peptide. 
     
     
         22 . The fluorescent polymer dot according to any one of  claims 1  to  21 , wherein the weight ratio of fluorescent conjugated polymer to amphiphilic molecule is in the range of 25% to 75%. 
     
     
         23 . The fluorescent polymer dot according to any one of  claims 1  to  22 , wherein the size of said dot is in the range of 30 nm to 50 nm. 
     
     
         24 . A method for preparing a fluorescent polymer dot, the method comprising the steps of:
 a) preparing a mixture of fluorescent conjugated polymer and amphiphilic molecule in an aprotic solvent; and   b) adding said mixture to a protic solvent, to form the polymer dot comprising one or more fluorescent conjugated polymers forming a hydrophobic core and one or more amphiphilic molecules, each amphiphilic molecule comprising a hydrophobic end embedded in the core and a hydrophilic end that forms a hydrophilic shell surrounding the core, wherein said hydrophobic end comprises one or more aliphatic chain moieties.   
     
     
         25 . The method according to  claim 24 , wherein the aprotic solvent is tetrahydrofuran or acetone. 
     
     
         26 . The method according to any one of  claims 24  to  25 , wherein the protic solvent is water. 
     
     
         27 . The method according to any one of  claims 24  to  26 , further comprising the step of controlling the size of the fluorescent polymer dot produced, the step comprising modifying the initial concentration of fluorescent conjugated polymer in aprotic solvent. 
     
     
         28 . The method according to  claim 27 , wherein the initial concentration of fluorescent conjugated polymer in aprotic solvent is in the range of 0.05 mg/mL to 1.50 mg/mL. 
     
     
         29 . The method according to any one of  claims 24  to  28 , wherein the amphiphilic molecule is attached to one or more functional groups. 
     
     
         30 . The method according to any one of  claims 24  to  29 , further comprising the step of conjugating a biological molecule to the surface of the fluorescent polymer dot. 
     
     
         31 . The method according to  claim 30 , wherein the biological molecule is conjugated to the fluorescent polymer dot via the one or more functional groups. 
     
     
         32 . A method of detecting a target molecule in a biological sample, the method comprising contacting a biological sample with a fluorescent polymer dot as claimed in any one of  claims 1  to  23 .